In an imaging system, adequate and appropriate illumination of the object to be imaged is essential. There must be enough light provided to the object to permit a viewer or detector to discern features of the object in the image thereof. In addition, the manner in which the light is provided to the object makes a difference in what features can be detected and the contrast with which they are imaged.
The way in which illumination is provided is particularly important in a microscope. If the object is opaque, it must be illuminated so that the light used to form an image of the object is radiated from the same side of the object on which light illuminates the object. This type of illumination is known primarily as epi-illumination. In epi-illumination the light radiated from an object may be in the form of reflection, in which case the illumination light is modulated upon reflection from the object, or it may be in the form of fluorescence, in which case the illumination light induces fluorescent emission by the object at a different wavelength from the illumination light, as determined by the fluorescence characteristics of the object. The latter case is known as epi-fluorescence. The term “radiated” is used throughout this specification and the claims hereof to include reflection, scattering and fluorescence.
One type of epi-illumination is critical illumination. In this case, the light source is imaged into the object plane. This provides efficient illumination and a short illumination system, but requires that the light source provide uniform radiance. The light source is ordinarily disposed actually or virtually on the optical axis of the imaging lens.
In the foregoing it is assumed that the entire field of view of the imaging lens is simultaneously imaged. However, in a confocal microscope only discrete points in object space are imaged. This is accomplished by placing one or more “pinhole” stops at the image plane of the microscope matched to corresponding discrete points in the object plane, and scanning the object laterally, either by moving the object or the microscope, or moving the scanning the beam through the microscope using, for example, scan mirrors. The light passed by the pinhole is detected and related to the object position as the scan occurs, and the output of the detector is used to produce an image of the object as a whole. In this case, light from the light source is focused to the point on the object plane that is currently imaged. This is typically accomplished by placing a beam splitter between the imaging lens and the image plane so as to pass image light to the image plane while reflecting source light from a virtual image plane created by the beam splitter along the optical axis of the microscope toward the object plane.
In classic optical instruments employing critical illumination, the image is detected by the human eye. In many modern optical instruments, the image is detected by a photo-sensitive device, typically an array of photodetectors. In confocal microscopy, the image is necessarily detected by some storage means. While the use of electronic image detection offers electronic capture of an image and the possibility of reducing the size of an imaging system, effective, compact epi-illumination has remained a challenge.
The recent development of array microscopes, also known as miniaturized microscope arrays, presents new challenges for illumination. In array microscopes a plurality of laterally-distributed optical imaging elements having respective longitudinal optical axes are configured to image respective sections of an object and disposed with respect to a common object plane so as to produce at respective image planes respective images of the respective sections. The individual lenses of this array are formed of small optical elements, or “lenslets,” that place severe constraints on providing illumination. Indeed, the multiplicity of lenslets arranged in an array and the small dimensions of the array suggest that prior art epi-illumination techniques cannot be used. Yet, a principal application for array microscopes is to image specimens, such as biological microarrays for protein analysis that are sufficiently opaque that dia-illumination cannot be used effectively.
Accordingly, there is a need for novel systems and methods for providing critical illumination in epi-illumination imaging systems employing electronic image detection.